Análises dos mecanismos imunopatológicos e moleculares envolvidos no processo de citoaderência de Plasmodium vivax

Orientadores: Fabio Trindade Maranhão Costa, Letusa AlbrechtTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Plasmodium vivax é o parasita causador malária humana mais prevalente, disseminado e negligenciado, colocando todos os anos bilhões de pessoas em risco de infeção, acarretando sérios problemas de saúde e econômicos. A emergência de resistência a antimaláricos e complicações clínicas graves são preocupantes. Pouco se sabe sobre os mecanismos envolvidos nas características patogénicas da biologia do negligenciado P. vivax. A impossibilidade de executar a cultura in vitro de isolados a longo prazo, limita os pesquisadores ao estudo da sua biologia no espaço e tempo, restringindo o trabalho experimental a áreas endêmicas de malária vivax, onde a execução bem-sucedida de aplicações ômicas é desafiadora. A capacidade de P. vivax remodelar a membrana dos reticulócitos do hospedeiro e promover a sua adesividade foi já demostrada, dendo um mecanismo importante de evasão ao sistema imunitário humano. Estudos funcionais têm reportado que a adesão de reticulócitos infectados por P. vivax (RTi-Pv) a células endoteliais do hospedeiro, apesar de em menor número, é forte e estável, como o verificado para eritrócitos infectados por P. falciparum. Também foi observado que a adesão de eritrócitos não infectados a RTi-Pv é forte e resulta na formação estável de rosetas, que apresenta uma maior taxa em malária vivax do que falciparum. Mais recentemente, foi publicado que existe uma correlação entre a formação de rosetas e a deformabilidade de RTi-Pv, na qual RTi-Pv que formam rosetas são significativamente mais rígidos. Estágios maduros de P. vivax (esquizontes) têm elevada capacidade de aderir relativa a outros estágios assexuais do parasita, em ambos fenótipos de citoadesão e roseteamento. A menor proporção de esquizontes na circulação sanguínea periférica de pacientes sugere que os parasitas podem sequestrar no endotélio vascular do hospedeiro. Os RTi-Pv que formam rosetas poderão ser a causa desta baixa taxa de esquizontes circulantes no sangue dos pacientes, contribuindo para o fenómeno de sequestro parasitário na microvasculatura e/ou baço do hospedeiro, e consequentemente contribuindo para características reopatológicas da malária vivax. Autópsias de pacientes com malária vivax mostraram a acumulação de RTi-Pv nos pulmões, baço, fígado e medula óssea. Ainda, foi demonstrado que a parasitemia subestima a biomassa total parasitária, que é elevada em malária vivax severa, portanto, capaz de mediar a inflamação sistémica da patologia. O objetivo deste estudo é a compreensão dos mecanismos moleculares envolvidos nos fenótipos adesivos, identificando proteínas, principalmente ligantes parasitários, que possam ser importantes na capacidade de aderência de P. vivax. Usando RNA-seq em conjunto com o enriquecimento, maturação ex vivo e ensaio funcional de adesão com amostras clínicas de P. vivax, foi sequenciado o transcriptoma de populações de parasitas com características adesivas distintas. Os nossos perfis de expressão mostram a importância de diferentes grupos de proteínas membranares ou associadas a membranas, com propriedades de adesinas, tais como proteínas Plasmodium Interspersed repeats (PIR) e Plasmodium Helical Interspersed SubTelomeric (PHIST), que podem ter um papel importante no fenótipo adesivo de P. vivax. Dentro deste grupo de proteínas diferencialmente expressas foi verificado que muitas são tradicionalmente produzidas por parasitas em fase sexuada, sugerindo a importância da formação de rosetas por gametócitos de P. vivax. Adicionalmente, análise do perfil de expressão gênica humano permitiu a identificação de genes diferencialmente expressos associados a vias de fagocitose. Estes dados sugerem fortemente que o fenótipo de roseteamento pode impedir a fagocitose do parasita por leucócitos como resposta do sistema imune humano à infeção. Os resultados obtidos refletem as características patogénicas de populações brasileiras circulantes de P. vivax, principalmente no que diz respeito à sua capacidade de aderência como principal fonte das manifestações clínicas severas reportadas. Para além disso, esperamos que estes dados abram ainda mais as investigações sobre a biologia deste parasita apicomplexo, ajudando no desenho de vacinas e na descoberta de novos antimaláricos, promovendo o sucesso na eliminação da malária vivax no futuro.Abstract: Plasmodium vivax is the most prevalent, widespread and neglected human malaria parasite, currently placing billions of people at risk of infection, thus imposing major health and economic burdens. Worldwide, anti-malarial drug resistance emergence and severe clinical complications are of great concern. The mechanisms underlying the pathobiology of the neglected P. vivax are still little known. The lack of a reliable in vitro P. vivax long-term culture restricts its biology study in place and time, relegating researchers to work in malaria endemic field conditions, where successful omics applications are very challenging. The capacity of P. vivax to remodel host reticulocyte membrane and promote adhesivity has been demonstrated, which is an important mechanism for host immune evasion. Functional studies have already reported that adhesion of P. vivax infected red blood cells (PvIRBCs) to the host endothelial cells, although in considerably lower rates, is as strong and stable as the verified for P. falciparum infections. Also, it has been reported adhesion of normocytes to the PvIRBCs is strong and results in stable rosette formation, which shows higher rates in vivax compared to falciparum malaria. More recently, it was reported that there is a correlation between rosette formation and altered membrane deformability of PvIRBCs, where the rosette-forming PvIRBCs are significantly more stiff and rigid than their non-rosetting equals. Mature staged parasites (schizonts) show a higher capacity for adherence than other asexual parasite stages both in cytoadherence and rosetting. The lower proportion of schizonts observed on the peripheral blood circulation of patients suggests that parasites could be sequestered on the host vascular endothelium. Rosette-forming PvIRBCs may also be the cause for this lower rate of schizonts in the patients’ blood, contributing for parasite sequestration phenomena in the host microvasculature and/or spleen, and consequently, the rheopathological characteristics present in vivax malaria disease. Vivax malaria patient autopsies have shown accumulation of PvIRBCs in the lungs, spleen, liver and bone marrow. Additionally, it has been demonstrated that parasitemia underestimates total parasite biomass, which is greater in severe vivax malaria patients, and thus, capable of mediating systemic inflammatory pathology. In this study, we aimed to understand the molecular mechanisms behind adherence phenotypes by identifying proteins, especially parasitic ligands, which might be important in P. vivax adhesion capacity. Using RNA-seq coupled with parasite field sample enrichment, ex vivo maturation and cytoadherence assays, we have sequenced the whole transcriptome of parasite populations with distinct adhesive characteristics. Our expression profiles brings out the importance of membrane and membrane-associated proteins, with adhesin or adhesin-like properties, such as Plasmodium Interspersed repeats (PIR) and Plasmodium Helical Interspersed SubTelomeric (PHIST) proteins, which might pay a role in adherence phenotypes. Within those protein groups, we found a percentage of differentially expressed genes that traditionally are more expressed in sexual rather than asexual parasite stages, suggesting the relevance of rosette formation by P. vivax gametocytes. Importantly, we found host immune-related differentially expressed genes, of which several are associated with the human phagocytosis pathways. These data strongly suggest that rosetting can hamper leukocyte phagocytosis host immune response, as an effective mechanism of P. vivax immune evasion adaptation. Our results reflect the pathobiology of circulating Brazilian P. vivax populations, principally concerning its adhesive capacity as a possible source of the severe clinical manifestations reported. Furthermore, we hope that such achievements will further enable the investigations on the biology of P. vivax apicomplexan parasite, impacting considerably in vaccine and drug design, ultimately helping us achieve the future elimination of vivax malaria.DoutoradoImunologiaDoutora em Genética e Biologia Molecular2013/20509-5FAPES

Potential for adaptation overrides cost of resistance

The deposited article is a post-print version and has been submitted to peer review.This deposit is composed by the main article plus the supplementary materials of the publication.This work is licensed under the Creative Commons Attribution-NonCommercial 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/To investigate the cost of antibiotic resistance versus the potential for resistant clones to adapt in maintaining polymorphism for resistance. Materials & methods: Experimental evolution of Escherichia coli carrying different resistance alleles was performed under an environment devoid of antibiotics and evolutionary parameters estimated from their frequencies along time. Results & conclusion: Costly resistance mutations were found to coexist with lower cost resistances for hundreds of generations, contrary to the hypothesis that the cost of a resistance dictates its extinction. Estimated evolutionary parameters for the different resistance backgrounds suggest a higher adaptive potential of clones with costly antibiotic resistance mutations, overriding their initial cost of resistance and allowing their maintenance in the absence of drugs.The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement no 260421 – ECOADAPT, and PTDC/BIA-EVF/114622/2009, financed by Fundação para a Ciência e Tecnologia (FCT). I Gordo acknowledges the salary support of LAO/ITQB & FCT. JM de Sousa acknowledges the scholarship SFRH/BD/89151/2012, from FCT.info:eu-repo/semantics/publishedVersio

A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of the SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the Escherichia coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor by a robust growth increase, discernible down to 1 mM. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARSCoV-2 main protease or even for other proteases

Micrornas In The Host-apicomplexan Parasites Interactions: A Review Of Immunopathological Aspects.

MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, have been detected in a variety of organisms ranging from ancient unicellular eukaryotes to mammals. They have been associated with numerous molecular mechanisms involving developmental, physiological and pathological changes of cells and tissues. Despite the fact that miRNA-silencing mechanisms appear to be absent in some Apicomplexan species, an increasing number of studies have reported a role for miRNAs in host-parasite interactions. Host miRNA expression can change following parasite infection and the consequences can lead, for instance, to parasite clearance. In this context, the immune system signaling appears to have a crucial role

MicroRNAs in the host-apicomplexan parasites interactions: a review of immunopathological aspects

MicroRNAs (miRNAs), a class of small non-coding regulatory RNAs, have been detected in a variety of organisms ranging from ancient unicellular eukaryotes to mammals. They have been associated with numerous molecular mechanisms involving developmental, physiological and pathological changes of cells and tissues. Despite the fact that miRNA-silencing mechanisms appear to be absent in some Apicomplexan species, an increasing number of studies have reported a role for miRNAs in host-parasite interactions. Host miRNA expression can change following parasite infection and the consequences can lead, for instance, to parasite clearance. In this context, the immune system signaling appears to have a crucial role6CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2012/16525-

Plasmodium vivax Biology: Insights Provided by Genomics, Transcriptomics and Proteomics

During the last decade, the vast omics field has revolutionized biological research, especially the genomics, transcriptomics and proteomics branches, as technological tools become available to the field researcher and allow difficult question-driven studies to be addressed. Parasitology has greatly benefited from next generation sequencing (NGS) projects, which have resulted in a broadened comprehension of basic parasite molecular biology, ecology and epidemiology. Malariology is one example where application of this technology has greatly contributed to a better understanding of Plasmodium spp. biology and host-parasite interactions. Among the several parasite species that cause human malaria, the neglected Plasmodium vivax presents great research challenges, as in vitro culturing is not yet feasible and functional assays are heavily limited. Therefore, there are gaps in our P. vivax biology knowledge that affect decisions for control policies aiming to eradicate vivax malaria in the near future. In this review, we provide a snapshot of key discoveries already achieved in P. vivax sequencing projects, focusing on developments, hurdles, and limitations currently faced by the research community, as well as perspectives on future vivax malaria research

A new β-hydroxydihydrochalcone from Tephrosia uniflora, and the revision of three β-hydroxydihydrochalcones to flavanones

The CH2Cl2/MeOH (1:1) extract of the stems of Tephrosia uniflora yielded the new β-hydroxydihydrochalcone (S)-elatadihydrochalcone-2'-methyl ether (1) along with the three known compounds elongatin (2), (S)-elatadihydrochalcone (3), and tephrosin (4). The structures were elucidated by NMR spectroscopic and mass spectrometric data analyses. Elongatin (2) showed moderate antibacterial activity (EC50 of 25.3 μM and EC90 of 32.8 μM) against the Gram-positive bacterium Bacilus subtilis, and comparable toxicity against the MCF-7 human breast cancer cell line (EC50 of 41.3 μM). Based on the comparison of literature and predicted NMR data with that obtained experimentally, we propose the revision of the structures of three β-hydroxydihydrochalcones to flavanones

Macrocyclic Pyrrolizidine Alkaloids and Silphiperfolanol Angelate Esters from Solanecio mannii

Three new compounds, the silphiperfolanol angelate ester umutagarananol (1), the macrocyclic pyrrolizidine alkaloids umutagarinine A and B (2–3), and five known secondary metabolites (4–8) were isolated from the CH2Cl2−MeOH (1 : 1) extract of the roots and the stem bark of Solanecio mannii (Hook. f.) (Asteraceae). The isolated compounds were characterized by NMR and IR spectroscopic, and mass spectrometric analyses, whereas the relative stereochemistry of 4 was established by NAMFIS-based combined computational and solution NMR analysis. Synthetic modification of 5 provided two new compounds, 2-angeloyloxy-4,8-epoxypresilphiperfolane (9) and 2-angeloyloxy-4,8-epoxypresilphi-perfolane (10). The crude extracts and the isolated constituents showed weak antibacterial activities (EC50 0.7–13.3 mM) against the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. Compounds 2, 3 and 4 exhibited strong cytotoxicity against MCF-7 human breast cancer cells, with EC50 values of 35.6, 21.7 and 12.5 μM, respectively

Development of Dicationic Bisguanidine-Arylfuran Derivatives as Potent Agents against Gram-Negative Bacteria

Antibiotic resistance among bacteria is a growing global challenge. A major reason for this is the limited progress in developing new classes of antibiotics active against Gram-negative bacteria. Here, we investigate the antibacterial activity of a dicationic bisguanidine-arylfuran, originally developed as an antitrypanosomal agent, and new derivatives thereof. The compounds showed good activity (EC50 2–20 µM) against antibiotic-resistant isolates of the Gram-negative members of the ESKAPE group (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli with different antibiotic susceptibility patterns, including ESBL isolates. Cytotoxicity was moderate, and several of the new derivatives were less cytotoxic than the lead molecule, offering better selectivity indices (40–80 for several ESKAPE isolates). The molecular mechanism for the antibacterial activity of these molecules is unknown, but sensitivity profiling against human ESKAPE isolates and E. coli collections with known susceptibility patterns against established antibiotics indicates that it is distinct from lactam and quinolone antibiotics

Benzo[b]naphtho[2,1-d]furans and 2-Phenylnaphthalenes from Streblus usambarensis

Three new benzo[b]naphtho[2,1-d]furans, usambarins A–C (1–3), five new 2-phenylnaphthalenes, usambarins D–H (4–8), a new flavan (9), and a new phenyl-1-benzoxepin (10) as well as two known compounds (11 and 12) were isolated from the extract of the stem and roots of Streblus usambarensis (Moraceae). The structures were deduced using NMR spectroscopic and mass spectrometric analyses, and those of compounds 1 and 4 were confirmed by X-ray crystallography. Usambarin D (4) demonstrated moderate antibacterial activity (MIC 9.0 μM) against Bacillus subtilis, while none of the tested compounds were effective against Escherichia coli